Chicken leukemia inhibitory factor (LIF) and gene thereof

ABSTRACT

The present invention is to provide a chicken LIF gene. Based on this genetic information, LIF protein derived from the chicken can be stably supplied and it solves the problems of the creation of transgenic chickens in the past. In addition, the present invention provides not only transgenic chickens for testing purposes but also supplies the first practical transgenic stock animals. The present invention relates to leukemia inhibitory factor (LIF) shown in sequence No. 2, and the gene that encodes thereof shown in sequence No. 1, and a manufacturing method of chicken LIF. In addition, the present invention pertains to a differential preventer of the chicken differentiable cell, and a method of chicken differential prevention and a culturing method for the chicken differentiable cell using thereof, and a medium comprising thereof.

FIELD OF TECHNOLOGY

[0001] The present invention relates to a chicken leukemia inhibitoryfactor (hereinafter referred to as “LIF”) and the gene thereof, as wellas its manufacturing method. The chicken LIF of the present inventionhas a differentiation prevention function for a chicken differentiablecell such as a chicken's embryonic stem cell or embryonic reproductivecell, and is effective as an anti-differentiator while the chickendifferentiable cell is being cultured.

[0002] The present invention also relates to a culturing method for achicken differentiable cell using a chicken LIF, a culture medium, and acreation method for a transgenic chicken using said method, and atransgenic chicken created with said method.

BACKGROUND OF THE INVENTION

[0003] Recently, the creation of transgenic animals has broughtsignificant progress in a wide range of areas including medicine andbiology. The creation of animals that are introduced with specific genesor knocked-out specific genes has become an important tool to analyzethe functions of these genes. There have been two types of creationmethods for transgenic animals: the transgenic method in which a foreigngene is directly introduced to a fertilized egg, and the geneintroduction method that is carried out via an embryonic stem cell(hereinafter referred to as an ES cell). Currently, the introduction ofa gene to the ES cell has been widely used because the target gene canbe introduced or there is a knock-out from a designated genome locationusing the gene targeting method.

[0004] As described above, the creation of transgenic animals using theES cell by the gene targeting method has been widely carried out. Inthis method, the inhibition of the

[0005] As described above, the creation of transgenic animals using theES cell by the gene targeting method has been widely carried out. Inthis method, the inhibition of the differentiation during theengineering of the gene is the significant issue. To culture an ES cell,the preparation of a feeder cell or the use of an LIF as a factor toinhibit the differentiation is required. The LIF is a type of cytokinethat belongs to the IL-6 family. It is an essential material to maintainthe cell in an undifferentiated state (Smith A., et al., Nature, 336,688-690 (1988)).

[0006] A mouse LIF is an essential factor to create a transgenic mousethrough the gene targeting method, and the recombinant LIF derived fromthe mouse has been commercialized. The culturing of a mouse ES cell hasbeen realized using this factor only, however, the LIF has not beenestablished for other animals and the creation of a transgenic animalusing the gene targeting method has been difficult.

[0007] The creation of transgenic chickens has been attempted becauseits egg has a high productivity of proteins. The fertilized egg of thechicken is relatively easy to handle so that the introduction of geneshas few problems. However, in order to introduce a knock-out of the geneto or from the designated location, the creation of a transgenic chickenusing the gene targeting method is necessary.

[0008] Chickens and mice have different development patterns so that theES cell cannot be used. However, it is possible to separate and culturea cell equivalent to the ES cell from the developing embryo of achicken. Chickens have blastodermic cells and primordial reproductivecells that are equivalent to a mouse ES cell. Blastodermic cells aredeveloping reproductive cells that are equivalent to the mouse ES cell.The primordial reproductive cell is a cell that is going to bedifferentiated to be a reproductive cell, and the development of a stockcell that can be externally cultured has advanced. This stock cell iscalled an embryonic germ cell. The issue is how to perform the culturingwhile maintaining the undifferentiated state for either cell. In anyevent, currently no chicken LIF has been found so that a medium that isa combination of another mammal LIF and another cytokine has been used.For example, a study has been carried out to establish a culturingsystem for chicken ES cells and embryonic reproductive cells using arecombinant LIF derived from a mouse. However, its effect is low andcurrently no culturing system has been established.

[0009] The chicken embryo is easy to obtain and the operation for theembryo is simple so that it has been widely used as a study material inembryology. In addition, the establishment of a production technologyfor transgenic chickens has been desired because chickens are not onlyused for their meat but also they have a high productivity of protein intheir eggs, and are industrially important. However, a culturing systemfor the chicken ES cell and embryonic reproductive cell has not beenestablished so that we have not reached the level where an artificialmodification of the gene can be easily carried out (at the level of thetransgenic mouse). Thus, the gene/protein cloning of chicken LIF, whichis an essential factor for the culturing system is being quickened.Nevertheless, chickens are significantly different from other mammalsand even the fact as to whether an LIF exists or not has not beendetermined. As described above, the manufacturing of chicken LIF proteinhas been desired in order to create transgenic chickens.

SUMMARY OF THE INVENTION

[0010] The present invention has taken advantage of state-of-the-artmolecular biological techniques in order to solve these problems and toprovide a gene for a chicken LIF. If a stable supply of the LIF proteinderived from chickens is possible based on this genetic information, theproblem of the creation of a transgenic chicken in the past can beresolved.

[0011] In addition, the present invention mass produces the recombinantLIF protein based on the genetic information of the present invention,and a supply thereof as the medium reagent for the chicken ES cell andembryonic reproductive cell in order to create a transgenic chicken.Consequently, it allows the providing of not only transgenic chickensfor test purposes but also the first practical transgenic stock animals.

[0012] The present invention relates to a chicken LIF, and the gene thatencodes thereof, and a manufacturing method for chicken LIF.

[0013] The present invention also relates to a differential preventer ofa chicken differentiable cells comprising the chicken LIF, thedifferentiation prevention method of the chicken using thereof, aculturing method for the chicken differentiable cells, and the mediumcomprising thereof.

[0014] The present invention further relates to a method of creatingtransgenic chickens in which said differentiable cell is cultured in thepresence of a chicken LIF, and the transgenic chicken that is createdusing said method.

[0015] The inventors of the present invention stimulated chickenmonocytic cell stock (IN24) with a lipopolysaccharide (LPS), and usingthe subtraction method, the gene that excessively developed due to theLPS stimulation, was cloned. Additionally, a clone that is homological(approximately 30%) to the LIF derived from a mammal was obtained amongthese clones.

[0016] The cDNA base sequence of the obtained clone is shown in sequenceNo. 1 in the sequence table. This gene is comprised of 789 bases and itis assumed that it encodes the protein comprising 211 amino acids havingan interpretation area of 75th atg to 708th tga.

[0017] The sequence of chicken LIF that is comprised of the assumed 211amino acids is shown in sequence No. 2 in the sequence table. The oneletter code of the amino acid of this amino acid sequence is shown asfollows. MRLIPAGVVP FVALLLLQRR PVSGRALLGT  30 SSACPTNGLC RANVLEQTRRQVALLNATAQ 60 DLFSLYLKCQ GEPFSSESDR LCSPSGIFFP 90 PFHVNRTTER KEVMVAMYKLFAFLNASLGN 120 ITRDQEELNP MAKELLNRLH NTTKTTRGLI 150 SNLTCLLCKHYNIFQVDVSY GESSKDKSAF 180 KKKQQGCQVL RKYVQVIAQA ARVLLPHLSP 210 A 211

[0018] In addition, the base sequence and amino acid (one letter code)of the obtained chicken LIF gene is shown in FIG. 1.

[0019] A comparison of the amino acid sequence assumed from the basesequence of the chicken LIF of the present invention with the amino acidsequence of the human LIF and mouse LIF is shown in FIG. 2. The blacktriangle in FIG. 2 shows the location of the cysteine and the whitetriangle shows the location where a saccharide can be added.

[0020] The amino acid sequence assumed from the base sequence of thechicken LIF of the present invention is homologous for less than 40%with the mouse LIF. Only a partial effect has been recognized with theculture system of the chicken ES cell and embryonic reproductive cellusing the recombinant mouse LIF. This supports the fact that the mouseLIF and chicken LIF have a low homologue. However, the location of thecysteine, which is an important amino acid that determines the structureof protein, was completely identical among the chicken, human and mouse.

BRIEF DESCRIPTION OF DRAWINGS

[0021]FIG. 1 shows a chicken amino acid sequence and the DNA basesequence of the present invention.

[0022]FIG. 2 shows a comparison of the chicken LIF amino acid sequencewith the human LIF and mouse LIF thereof.

[0023]FIG. 3 is a photo in place of a drawing that shows a selectedclone from the clones obtained by the subtraction method.

[0024]FIG. 4 shows the location of the primer and its sequence when theRACE method is applied to the clones obtained by the subtraction method.

[0025]FIG. 5 shows the schematic structure of a cDNA base sequence of achicken LIF of the present invention. The arrow shows the location ofthe primer of the RT-PCR method that was carried out in order to confirmthe sequence.

[0026]FIG. 6 is a photo in place of a drawing that shows the results ofa 502 bp fragment obtained by the RT-PCR method.

PREFERRED EMBODIMENTS OF THE INVENTION

[0027] The chicken LIF of the present invention is not limited to theone having the amino acid sequence shown in sequence No. 2 in thesequence table. As long as it is LIF active for a chicken, a portion ofthe amino acid shown in sequence No. 2 can be deleted, or substitutedwith another amino acid, and/or added with another amino acid.

[0028] There is no particular limitation on the number of amino acidsfor deletion, substitution or addition, however it is preferable for itto be half or less, which is 1 to 100 or 1 to 50 pieces.

[0029] The chicken LIF gene of the present invention is not limited tothose of them that have the base sequence shown in sequence No. 1 of thesequence table. As long as it encodes the protein that is LIF active forthe chicken as described above, a portion of the amino acid shown insequence No. 1 can be deleted, or substituted with another amino acid,and/or added with another amino acid. In addition, the chicken LIF geneof the present invention can be a double strand DNA or single strand DNAor RNA. Those of them that have a supplemental base for said chicken LIFof the present invention are included in the chicken LIF gene of thepresent invention. Furthermore, the chicken LIF gene of the presentinvention includes a base sequence that can be hybridized understringent conditions.

[0030] In addition, the present invention provides a fragment of achicken LIF gene of the present invention. The fragment of the chickenLIF gene of the present invention can be used as a probe to detect,identify or quantify the chicken LIF gene of the present invention, andas a primer in order to obtain the chicken LIF gene of the presentinvention.

[0031] As a fragment of the chicken LIF gene of the present invention,approximately 5 to 50 bases, 5 to 30 bases or 10 to 30 bases of thesequence from an arbitrary location can be used.

[0032] The manufacturing method of the chicken LIF of the presentinvention can be carried out using a well-known method using saidchicken LIF gene of the present invention. For example, a gene that hasthe base sequence shown in sequence No. 1 is combined into amanifestation vector and a phenotypic transformation of the host cell iscarried out. Additionally, it can be manufactured through the culturingof the phenotypically transformed host cell. A prokarytoic cell such asE. coli or an eukaryotic cell such as a cell from yeast or a mouse canbe used as the host cell.

[0033] Based on the chicken LIF gene of the present invention, arecombinant chicken LIF can be created using a prokarytoic cell or aeukaryotic cell, and consequently, the mass production of the chickenLIF necessary to create a transgenic chicken can be realized.

[0034] Examples of the chicken differentiable cell of the presentinvention are, chicken ES cells and embryonic reproductive cells.

[0035] When a chicken differentiable cell is cultured, the chicken LIFof the present invention can prevent the differentiation of said cell bybeing added in the culture system. Therefore the chicken LIF of thepresent invention can be used as the differentiation preventer of thechicken differentiable cell. Purified chicken LIF of the presentinvention can be individually used as the differentiation preventer ofthe present invention or it can be used in a mix with a carrier. Moreover, a medium to culture the chicken differentiable cells can becreated by mixing the LIF of the present invention with the medium inadvance.

[0036] Furthermore, the present invention provides a culturing methodfor the chicken differentiable cells using the LIF of the presentinvention. It provides a culturing method for a chicken differentiablecells by adding said differentiation preventer of the present inventioninto the culture system, or using said medium of the invention.

[0037] In addition, the present invention provides a creation method fora transgenic chicken and a transgenic chicken created using the methodhereof. An example of the desirable creation method of the transgenicchicken of the present invention is such that the differentiable cellssuch as the chicken ES cells and embryonic reproductive cells withmodified genetic information are modified and cultured thereof:preferably by introducing a foreign gene that can be developed at aspecific genome location through a gene targeting method; or through amodification in order to inhibit the development of a specific gene atspecific genome locations, for instance, an introduction of a foreigngene to destroy an Exon area such as a promoter area or a modificationof the gene to modify or add encoded amino acid. When chicken ES cellsor embryonic reproductive cells are used as the differentiable cells,normally, a chimera chicken is obtained. Additionally, the nextgeneration thereof becomes a transgenic chicken in which the target geneis introduced or knocked out.

[0038] The recombinant chicken LIF created based on the chicken LIF geneof the present invention is a discovery with hope in order to provide anestablished method for the creation of transgenic chickens. The use ofthis allows the maintenance of the subculture of the chicken ES cellsand embryonic reproductive cells in an undifferentiated state, which hasbeen impossible in the past, and also allows the in-vitro selection ofthe introduced gene, which has been difficult for chickens. In otherwords, all the problems regarding the creation of a transgenic chickenhave been resolved with the present invention.

[0039] In addition, when the culture system of these cells isestablished, the creation of a transgenic chicken will become evensimpler than a mouse, allowing the poultry industry to produce (producedas eggs) utility materials (medical drugs, reagents for clinical tests,etc.).

EMBODIMENTS

[0040] The present invention is described in detail using embodiments asfollows, however, the present invention is not limited to theseembodiments.

[0041] Embodiment 1 (Gene Cloning of the Chicken LIF)

[0042] mRNA was purified from chicken monocytic cell stock (IN24)stimulated with a lipopolysaccharide (LPS) and not stimulatedseparately, and then double strand cDNA was synthesized.

[0043] Using the cDNA obtained from these two types of cells, asubtraction method was carried out using the Clontech PCR-select cDNAsubtraction kit. The cDNA obtained was cloned to the pGEM-T vector.These clones were phenotypically transformed to JM109 E-coli andcultured on an agar medium and then the developed colony was selectedusing the PCR method. It was selected such that the PCR was carried outusing the pUC-M13 forward primer and reverse primer in the pGEM-T vectorbase sequence and only those clones with cDNA inserted in the vectorwere selected. The results are shown in FIG. 3 as a photo in place of adrawing.

[0044] Finally, 122 clones were selected and the plasmid of these cloneswas created. Then using an auto-sequencer, the base sequence of eachclone was determined.

[0045] Each of the determined base sequences were checked against adatabase, and then it was found that one of the clones is a clone thatis homologous with a mammalian (cows, pigs, etc.) derived LIF.

[0046] As described above, we determined that the obtained clone is apart of the gene (259 bp) that encodes the chicken LIF. Based on thisbase sequence of the clone, the cloning of the complete chicken LIF wascarried out by the RACE method using the Clontech smart-RACE kit.

[0047] Based on the obtained chicken LIF gene base sequence, at thelocation shown in FIG. 4, primers for 3′ and 5′ were prepared and a PCRwas carried out between the adaptor sequences. The top line of FIG. 4 isa schematic view of the 259 bp sequence obtained as above, and thebottom line shows its specific sequence.

[0048] As a result, 789 bp was determined including the entiretranslated area.

[0049] The determined base sequence was confirmed from the fragmentobtained through the RT-PCR method using the new primer shown in FIG. 5.The square-framed portion in FIG. 5 shows the translated area and theshadowed portion shows the 259 bp portion obtained through saidsubtraction method. The arrow in the bottom line in FIG. 5 shows theprimer portion of the RT-PCR.

[0050] The results of electrophoresis of the 502 bp fragment amplifiedby the RT-PCR method are shown in FIG. 6 as a photo in place of adrawing. The lane M indicates the marker and the black triangle showsthe location of the 502 bp fragment.

[0051] The base sequence and amino acid (one letter code) of theobtained chicken LIF gene is shown in FIG. 1. Its base sequence is shownin sequence No. 1 and the amino acid sequence is shown in sequence No.2.

[0052] The present invention is to provide a chicken LIF and the genethereof that is necessary to create a chicken with introduced foreigngenes (transgenic chicken) for the first time. The present invention ison the frontier of the creation of a chicken with introduced foreigngenes (transgenic chicken) and in addition, it is expected to lead tothe mass production of utility materials such as medical drugs. So far,the progress of studies and the practical use of the creation of atransgenic chicken have been extremely delayed because a chicken LIF hasbeen unknown, however, the providing of the chicken LIF of the presentinvention allows practical use to the same extent as that for a mouse.

[0053] It is easy to obtain the embryo of a chicken and it has goodoperability. The chicken LIF of the present invention allows theestablishment of a culturing system of chicken ES cells, allowing thecreation of transgenic chickens, which is even easier than that for amouse. This is not just at the laboratory level but also it allows theapplication to breeding in the poultry industry. In addition, becausethe chicken egg has a high productivity of protein, it allows thepractical manufacturing of utility materials (medical drugs, clinicaltest reagents, etc.). The present invention opens up the road for thefirst time to a supply of practical level transgenic stock animals.

[0054] The disclosure of Japanese Patent Application No. 2001-171993filed Jun. 7, 2001 including specification, drawings and claims areherein incorporated by reference in their entirety.

[0055] Although only some exemplary embodiments of this invention havebeen described in detail above, those skilled in the art will readilyappreciated that many modifications are possible in the exemplaryembodiments without materially departing from the novel teachings andadvantages of this invention. Accordingly, all such modifications areintended to be included within the scope of this invention.

1 7 1 211 PRT chicken 1 Met Arg Leu Ile Pro Ala Gly Val Val Pro Phe ValAla Leu Leu Leu 1 5 10 15 Leu Gln Arg Arg Pro Val Ser Gly Arg Ala LeuLeu Gly Thr Ser Ser 20 25 30 Ala Cys Pro Thr Asn Gly Leu Cys Arg Ala AsnVal Leu Glu Gln Thr 35 40 45 Arg Arg Gln Val Ala Leu Leu Asn Ala Thr AlaGln Asp Leu Phe Ser 50 55 60 Leu Tyr Leu Lys Cys Gln Gly Glu Pro Phe SerSer Glu Ser Asp Arg 65 70 75 80 Leu Cys Ser Pro Ser Gly Ile Phe Phe ProPro Phe His Val Asn Arg 85 90 95 Thr Thr Glu Arg Lys Glu Val Met Val AlaMet Tyr Lys Leu Phe Ala 100 105 110 Phe Leu Asn Ala Ser Leu Gly Asn IleThr Arg Asp Gln Glu Glu Leu 115 120 125 Asn Pro Met Ala Lys Glu Leu LeuAsn Arg Leu His Asn Thr Thr Lys 130 135 140 Thr Thr Arg Gly Leu Ile SerAsn Leu Thr Cys Leu Leu Cys Lys His 145 150 155 160 Tyr Asn Ile Phe GlnVal Asp Val Ser Tyr Gly Glu Ser Ser Lys Asp 165 170 175 Lys Ser Ala PheLys Lys Lys Gln Gln Gly Cys Gln Val Leu Arg Lys 180 185 190 Tyr Val GlnVal Ile Ala Gln Ala Ala Arg Val Leu Leu Pro His Leu 195 200 205 Ser ProAla 210 2 789 DNA Chicken 2 gcgggggaac atgaatttct gaaaaccctc acacgccgcccgtctgcgct cggctctccg 60 ggacggcgct caccatgagg ctcatccccg caggtgtcgtgcccttcgtg gccctgctgc 120 tgctgcagag gaggccggtg tccgggcggg cgctgctggggacgagctct gcgtgtccca 180 ccaacgggct gtgccgggcc aatgtcctgg agcagacccgcaggcaggtc gcactgctca 240 acgccaccgc gcaggacctc ttcagcctct atctgaagtgccagggagag ccgttcagca 300 gcgagagcga ccgcctctgc agccccagtg gcatcttcttcccccccttc cacgtcaacc 360 ggaccaccga gaggaaggag gtgatggtgg ccatgtacaagctcttcgcc ttcctcaacg 420 cctcactggg gaacatcacc cgcgaccagg aggagctcaaccccatggcc aaggagctcc 480 tcaaccgcct ccacaacacc accaaaacca cgcggggcctcatctccaac ctcacctgcc 540 tgctctgcaa gcactacaac atcttccagg tggacgtgagctacggggag agcagcaagg 600 acaagagcgc cttcaagaag aagcagcagg gctgccaggtgctcaggaag tacgtgcagg 660 tcatcgccca ggctgctcgt gtcctcctac ctcacctcagccccgcgtga gccccggccc 720 ccgcgccacg ctaccaccgg gacagcggac atctccttgcacccgtctca gagatgggca 780 cggcgaggc 789 3 22 DNA Artificial SequencePrimer 3 agagccgttc agcagcgaga gc 22 4 22 DNA Artificial Sequence Primer4 gcctcgccgt gcccatctct ga 22 5 259 DNA chicken 5 acaagctctt cgccttcctcaacgcctcac tggggaacat cacccgcgac caggaggagc 60 tcaaccccat ggccaaggagctcctcaacc gcctccacaa caccaccaaa accacgcggg 120 gcctcatctc caacctcacctgcctgctct gcaagcacta caacatcttc caggtggacg 180 tgagctacgg ggagagcagcaaggacaaga gcgccttcaa gaagaagcag cagggctgcc 240 aggtgctcag gaagtacct 2596 201 PRT Homo Sapien 6 Met Lys Val Leu Ala Ala Gly Val Val Pro Leu LeuLeu Val Leu His 1 5 10 15 Trp Lys His Gly Ala Gly Ser Pro Leu Pro IleThr Pro Val Asn Ala 20 25 30 Thr Cys Ala Ile Arg His Pro Cys His Asn AsnLeu Met Gln Ile Arg 35 40 45 Ser Gln Leu Ala Gln Leu Asn Gly Ser Ala AsnAla Leu Phe Ile Leu 50 55 60 Tyr Tyr Thr Ala Gln Gly Glu Pro Phe Pro AsnAsn Leu Asp Lys Leu 65 70 75 80 Cys Gly Pro Asn Val Thr Asp Phe Pro ProPhe His Ala Asn Gly Thr 85 90 95 Glu Lys Ala Lys Leu Val Glu Leu Tyr ArgIle Val Val Tyr Leu Gly 100 105 110 Thr Ser Leu Gly Asn Ile Thr Arg AspGln Lys Ile Leu Asn Pro Ser 115 120 125 Ala Leu Ser Leu His Ser Lys LeuAsn Ala Thr Ala Asp Ile Leu Arg 130 135 140 Gly Leu Leu Ser Asn Val LeuCys Arg Leu Cys Ser Lys Tyr His Val 145 150 155 160 Gly His Val Asp ValThr Tyr Gly Pro Asp Thr Ser Gly Lys Asp Val 165 170 175 Phe Gln Lys LysLys Leu Gly Cys Gln Leu Leu Gly Lys Tyr Lys Gln 180 185 190 Ile Ile AlaVal Leu Ala Gln Ala Phe 195 200 7 203 PRT Mouse 7 Met Lys Val Leu AlaAla Gly Ile Val Pro Leu Leu Leu Leu Val Leu 1 5 10 15 His Trp Lys HisGly Ala Gly Ser Pro Leu Pro Ile Thr Pro Val Asn 20 25 30 Ala Thr Cys AlaArg Ile His Pro Cys His Gly Asn Leu Met Asn Gln 35 40 45 Ile Lys Asn GlnLeu Ala Gln Leu Asn Gly Ser Ala Asn Ala Leu Phe 50 55 60 Ile Ser Tyr TyrThr Ala Gln Gly Glu Pro Phe Pro Asn Asn Val Glu 65 70 75 80 Lys Leu CysAla Pro Asn Met Thr Asp Phe Pro Ser Phe His Gly Asn 85 90 95 Gly Thr GluLys Thr Lys Leu Val Glu Leu Tyr Arg Met Val Ala Tyr 100 105 110 Leu SerAla Ser Leu Thr Asn Ile Thr Arg Asp Gln Lys Val Leu Asn 115 120 125 ProThr Ala Val Ser Leu Gln Val Lys Leu Asn Ala Thr Ile Asp Val 130 135 140Met Arg Gly Leu Leu Ser Asn Val Leu Cys Arg Leu Cys Asn Lys Tyr 145 150155 160 Arg Val Gly His Val Asp Val Pro Pro Val Pro Asp His Ser Asp Lys165 170 175 Glu Ala Phe Gln Arg Lys Lys Leu Gly Cys Gln Leu Leu Gly ThrTyr 180 185 190 Lys Gln Val Ile Ser Val Val Val Gln Ala Phe 195 200

We claim:
 1. A chicken leukemia inhibitory factor (LIF).
 2. A chickenleukemia inhibitory factor (LIF) in accordance with claim 1, wherein aportion of the amino acid sequence shown in sequence No. 2 of thesequence table or the amino acid shown in sequence No. 2 is deleted, andsubstituted with another amino acid and/or another amino acid is added,and the chicken LIF is active.
 3. A gene that encodes chicken leukemiainhibitory factor (LIF).
 4. A gene in accordance with claim 3 whereinthe gene that encodes the chicken leukemia inhibitory factor (LIF) iscomprised of a base sequence shown in sequence No. 1 of the sequencetable, or the base sequence shown in sequence No. 1 and the basesequence obtained through hybridization under stringent condition.
 5. Anoligonucleotide comprised of 5 to 50 bases at an arbitrary location ofthe gene that encodes the chicken leukemia inhibitory factor (LIF). 6.An oligonucleaotide wherein the bases are 10 to 30 in accordance withclaim
 5. 7. A manufacturing method of chicken leukemia inhibitory factor(LIF) using a gene that encodes chicken leukemia inhibitory factor(LIF).
 8. A vector that comprises a gene that encodes chicken leukemiainhibitory factor (LIF).
 9. A vector according to claim 8, wherein thevector is a development vector.
 10. A differentiation preventer of achicken differentiable cell comprising a chicken leukemia inhibitoryfactor (LIF).
 11. A differentiation preventer according to claim 10,wherein the chicken differentiable cell is a chicken embryonic stem cellor embryonic reproductive cell.
 12. A method of preventingdifferentiation of a chicken differentiable cell comprising a step ofadding the chicken leukemia inhibitory factor (LIF) into a medium of adifferentiable cell.
 13. The method of preventing differentiation of achicken differentiable cell according to claim 12, the differentiablecell is a chicken differentiable cell.
 14. The method in accordance withclaim 13, wherein the chicken differentiable cell is a chicken embryonicstem cell or embryonic reproductive cell.
 15. A medium to culture achicken differentiable cell that comprises leukemia inhibitory factor(LIF).
 16. The medium in accordance with claim 15 wherein the chickendifferentiable cell is a chicken embryonic stem cell or an embryonicreproductive cell.
 17. A method for making a transgenic chicken whereinthe method comprises steps of introducing a foreign gene into a chickendifferentiable cell, or modifying a predetermined gene in the chickendifferentiable cell to prevent development of a specific gene; andculturing the differentiable cell in a presence of chicken leukemiainhibitory factor (LIF).
 18. The method for making a transgenic chickenaccording to claim 17, wherein the chicken differentiable cell is achicken embryonic stem cell or an embryonic reproductive cell.
 19. Thetransgenic chicken created by the method in accordance with claim 17.20. The transgenic chicken created by the method in accordance withclaim 18.